This invention, in general, relates to polyphenolic products, particularly proanthocyanidins. The invention further relates to synthetic processes for preparing polyphenolic natural products and other related compounds.
Proanthocyanidins (nonhydrolyzable tannins) are a group of polyphenolic natural products, of current interest because of their numerous biological activities, their widespread occurrence in foodstuffs, and their resulting relevance for human health.
Proanthocyanidins are dimeric or oligomeric flavanoids which have one or several hydroxyl groups on their aromatic rings and often an additional hydroxyl group in the 3 position. Eleven different hydroxylation patterns of the A and B rings have been found in nature. Representative proanthocyanidins include:
The stereochemistry of the substituents on a polyphenol monomeric unit of a proanthocyanidin may be described in terms of their relative stereochemistry, xe2x80x9calpha/betaxe2x80x9d or xe2x80x9ccis/transxe2x80x9d. The term xe2x80x9calphaxe2x80x9d (xcex1) indicates that the substituent is oriented below the plane of the flavan ring, whereas, xe2x80x9cbetaxe2x80x9d (xcex2) indicates that the substituent is oriented above the plane of the ring. The term xe2x80x9ccisxe2x80x9d indicates that two substituents are oriented on the same face of the ring, whereas xe2x80x9ctransxe2x80x9d indicates that two substituents are oriented on opposite faces of the ring.
The isolation of pure proanthocyanidins from natural sources becomes increasingly difficult with increasing degree of oligomerization. Degradation by thiolysis permits identification of the underlying monomeric units but the tasks of elucidating the position and stereochemistry of the interflavan linkages is nontrivial. Both of these factors have resulted in few defined oligomers above the tetramer being described in the prior art. Proanthocyanidins and their parent monomers also occur naturally in the form of a variety of derivatives, for example, glycosides or esters with hydroxylated aromatic carboxylic acids, such as gallic or hexahydroxydiphenic acid.
Among the proanthocyanidins, two subtypes, the procyanidins (5,7,3xe2x80x2,4xe2x80x2-hydroxylation) and prodelphinidins (5,7,3xe2x80x2,4xe2x80x2,5xe2x80x2-hydroxylation), are widespread in human foodstuffs, e.g., cocoa. Cocoa procyanidins consist predominantly of epicatechin (the C-3 epimer of catechin) building blocks. Oligomers up to the size of the decamer have been identified. From the pentamer on, these oligomers exhibit growth inhibitory activity against various cancer cell lines. (Romanczk, L. J. Jr.; Hammerstone, J. F., Jr.; Buck, M. M. U.S. Pat. No. 5,554,645, Sep. 10, 1996.) Flavan-3-ols are biosynthetically derived from (2S)-phenylalanine via flavan-3,4-diols. These latter intermediates readily form a highly stabilized carbenium ion (or quinone methide) in position C-4 which attacks the A ring of a flavan-3-ol in what is essentially a Friedel-Crafts alkylation process, forming an interflavan bond. This process can be repeated once or several times, resulting in chain-type oligomers which together with the dimers are known as non-hydrolyzable tannins, condensed tannins, or proanthocyanidins. As one skilled in the art will realize, the structural complexity of these compounds rapidly increases with their chain length as a consequence of different hydroxylation patterns and C-3 stereochemistry in the monomer unit and different regio- and stereochemistries of the interflavan linkages, as well as additional structural modifications. In addition, chain branching may occur by alkylation of a monomer unit in both its 6- and 8-positions.
To prove definitively the structures assigned to the compounds purified from cocoa, comparisons must be made to epicatechin dimers and oligomers of defined structure prepared synthetically. Synthetic monomers, dimers and oligomers are useful to develop structure-activity relationships in various in vitro and ultimately in vivo models of anticancer activity.
The synthetic challenge posed by procyanidins is related to the difficulty in controlling the interflavan regio- and stereochemistry, as well as the sensitivity of the nonprotected compounds to acids, bases, and oxidizing agents. The condensation between flavan-3-ols and 4-substituted, electrophilic flavans has traditionally been performed without the use of phenol protecting groups in a mildly acidic medium or recently, with AgBF4 for benzylthio as the 4-substituent. The products are mixtures of regio- and sometimes stereoisomers, as well as higher oligomers despite the application of an excess of the nucleophilic building block. They have usually been separated by gel chromatography on Sephadex LH-20, a process that requires a considerable investment of time to develop for each particular separation task because of the unavailability of fast analytical tools such as HPLC columns or thin layer plates for this adsorbent. In addition, optically pure, nonprotected 4-substituted catechins and epicatechins are not readily available, being prepared by reduction of the expensive natural product, (+)-taxifolin (the 4-ketone) or by in situ degradation or thiolytic degradation of natural proanthocyanidin oligomic fractions for which commercial sources are difficult to identify or nonexistant.
It is therefore not surprising that prior art syntheses have used protected oligomeric procyanidins as building blocks. As an additional incentive, protection of the phenolic but not of the alcoholic hydroxyls would permit the regioselective elaboration of derivatives such as 3-esters and -glycosides, as has been done in the case of catechin using acetyl protecting groups. An interesting approach has been reported in which the 8-bromo derivative of 3-O-benzyl-5,7,3xe2x80x2,4xe2x80x2-tetra-O-methylcatechin was subjected to halogen-lithium exchange and reacted with an O-methylated 4-ketone, thus ensuring complete regiocontrol. However, the methyl O-blocking groups cannot be removed to obtain the free dimer. The remaining published work has made use of the above-described electrophilic substitution process with inclusion of phenol protecting groups on one or both of the reaction partners.
Our own previous work directed toward the synthesis of defined epicatechin oligomers used the TiCl4-mediated alkylation of 5,7,3xe2x80x2,4xe2x80x2-tetra-O-benzyl-(xe2x88x92)-epicatechin with 5,7,3xe2x80x2,4xe2x80x2-tetra-O-benzyl-4-(2-hydroxyethoxy)epicatechin. Besides higher oligomers, whose yields rapidly decrease with increased molecular mass, a single dimeric product having beta stereochemistry of the interflavan bond (a procyanidin B2 derivative) was obtained.
Until quite recently, the analytical methods employed for the assignment of interflavan bond regio- and stereochemistry in these compounds were not validated by an independent confirmation of the structure of any dimeric proanthocyanidin. The application of X-ray crystallography has been prevented by the poor crystallizability of proanthocyanidins and their derivatives. Assignments of stereochemistry on the basis of 1H NMR coupling constants and circular dichroism disregard the basic fact that the C rings are conformationally flexible. From a conservative point of view, postulates of specific conformations of flexible molecules, regardless of their source (intuitive or computational), cannot be considered a prudent approach to structure elucidation.
Advances in synthetic methodology, which have taken place after the isolation of numerous proanthocyanidins from natural sources, have now enabled one to obtain a definitive proof of the previously conjectured 4xcex2 stereochemistry in procyanidin B2. A differentially protected epicatechin dimer, correlated with procyanidin B2 through a common derivative, was subjected to a series of defunctionalization steps and finally degraded to (R)-(xe2x88x92)-2,4-diphenylbutyric acid, isolated as its benzhydryl ester. The sole remaining asymmetric center of this degradation product is directly derived from C-4 of the xe2x80x9ctopxe2x80x9d epicatechin moiety in procyanidin B2, and the sign of the optical rotation of the degradation product, the absolute configuration of which was established by X-ray crystallography, thus revealed the absolute configuration at C-4.
Those skilled in the art will recognize the importance of having an authentic sample of the opposite stereoisomer, now recognizable by default as epicatechin-4xcex1,8-epicatechin for comparison. Literature reports of proanthocyanidins for which simultaneously a 2,3-cis and a 3,4-cis relationship of their C-ring substituents has been postulated are scarce, and epicatechin-4xcex1,8-epicatechin has not been isolated from natural sources. In fact no stereoselective synthesis of any procyanidin containing a 4xcex1-linked unit has been reported to date.
It is a plausible assumption that, in the course of the formation of the 4xcex2,8-dimer, the 2-aryl group and the 3-oxygen cooperate in directing the approach of the flavan nucleophile to the presumed carbocationic intermediate towards its xcex2-face. Therefore, accessibility of the 4xcex1-stereoisomer by merely modifying the reaction conditions or attaching protecting groups to one or both of the 3-hydroxyl groups was deemed unlikely. Hence, there is a need for a new synthetic process for the preparation of epicatechins substituted at the 4xcex1 position. The instant disclosure is addressed to that unmet need.
The process of the present invention allows one to prepare unprotected epicatechin derivatives substituted at the 4xcex1 position, preferably with an aromatic radical. In one preferred embodiment, the dimer epicatechin-4xcex1,8-epicatechin is prepared. One advantage of the present invention is the formation of nonpolar intermediates which can be more readily separated at each step than polar, more sensitive final intermediates.
A crucial step in the present process is a transformation wherein a C-4-carbocation which has an aromatic unit already in place, is attacked by a hydride nucleophile from its xcex2-face, which forces the 4-aryl group to occupy the xcex1-face. The carbocation would be conveniently generated from a tertiary alcohol which, in turn, is accessible from an aryl organometallic reagent and a protected 4-keto-epicatechin.
This invention is directed to a process for preparing 40xcex1-aryl substituted epicatechins. The process comprises the steps of:
(A) forming a suitably protected epicatechin by protecting the C-3 hydroxyl group of 5,7,3xe2x80x2,4xe2x80x2-tetra-O-benzylepicatechin with a protecting group to produce a compound having the formula: 
where Bn is a benzyl group and R is a hydroxyl protecting group.
(B) oxidizing the 4-position of the protected epicatechin to produce a protected flavan-4-one having the formula: 
(C) contacting the flavan-4-one with a nucleophilic aryl organometallic reagent to produce a compound of formula: 
(D) deoxygenating the C-4 position stereoselectively to produce a compound of formula: 
(E) optionally deprotecting the C-3 hydroxyl group and then further optionally acylating the C-3 hydroxyl group with a suitable acylating agent and subsequently removing the benzyl groups; or
(A) optionally deprotecting the C-3 hydroxyl group and removing the benzyl groups to produce a free 4xcex1-aryl-epicatechin having the formula: 
Also provided is a process for the preparation of other derivatives of the 4xcex1-arylepicatechin by acylating the phenolic groups after removal of the benzyl groups.
In one aspect, the invention relates to a process for the preparation of epicatechin-epicatechin dimers where the nucleophilic aryl organometallic reagent is derived from a protected 8-bromoepicatechin or derivative thereof.
As used herein, the term epicatechin (an epimer of catechin) refers to a compound of formula: 
5,7,3xe2x80x2,4xe2x80x2-Tetra-O-benzylepicatechin refers to an epicatechin where the proton of each phenolic hydroxyl group is replaced by a benzyl group. Those skilled in the art of organic synthesis will recognize that there may be many methods of preparing tetra-O-benzylepicatechin. One particularly useful method of obtaining such a compound has been described by Txc3xcckmantel et al. J. Am. Chem. Soc. 1999, 121, 12073-12081.
As used herein, xe2x80x9carylxe2x80x9d means an aromatic hydrocarbon compound or heterocycle. The aryl group may be phenyl or substituted phenyl, wherein substituents are selected from the group consisting of halo, aryl, C1-C6-alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C3-C8 cycloalkyl, and C3-C8 cycloalkoxy provided that they are compatible with the formation of a nucleophilic organometallic reagent. The aryl group may be embedded in a ring-system of other aromatic or saturated hydrocarbon rings or heterocycles.
An important transformation in the present process is the protection of the hydroxyl group at the C-3 position of 5,7,3xe2x80x2,4xe2x80x2-tetra-O-benzylepicatechin. In one embodiment the protection involves alkylation with a benzyl halide, such as benzyl bromide to obtain a fully protected epicatechin. Typically, the reaction is conducted in the presence of a base, preferably a strong base such as an alkali metal hydride, dialkylamide, bis(trialkylsilyl)amide or hydroxide, more preferably an alkali metal hydride such as sodium hydride. The reaction is typically carried out in an a polar organic solvent. Those skilled in the art will be able to select a solvent that is compatible with a particular base. A preferred solvent may be acetonitrile, tetrahydrofuran (THF), N,N-dimethylformamide, a sulfoxide such as dimethylsulfoxide, orN-methylpyrrolidinone. A more preferred solvent is an amide such as N,N-dimethylformamide. A typical reaction temperature is between about 0xc2x0 C. and the reflux temperature of the solvent, preferably a temperature between 15xc2x0 C. and about 40xc2x0 C., more preferably about 23xc2x0 C. After addition of all the reagents, a reaction typically is stirred for a period between 15 minutes and 24 hours, preferably between 30 minutes and 1 hour.
In another embodiment, the protecting group used to protect the hydroxyl group at the C-3 position of 5,7,3xe2x80x2,4xe2x80x2-tetra-O-benzylepicatechin is orthogonal to a benzyl group. Typically the reaction involves O-silylation of the C-3 hydroxyl group to obtain a silyl ether. The silylating agent may be a silyl chloride. When the alkyl substituents on the silicon are tert-butyldimethyl, the silylating agent is tert-butyldimethylsilyl chloride, and the reaction is carried out in the presence of a mild base such as imidazole, or triethylamine with 4-(dimethylamino)pyridine. The reaction is carried out in an inert polar organic solvent, preferably N,N-dimethylformamide, dichloromethane, or THF. Typically the O-silylation reaction occurs at temperature between 0xc2x0 C. and about 40xc2x0 C., preferably at between about 15xc2x0 C. and about 30xc2x0 C., more preferably about 23xc2x0 C., for a period between about 1 and about 24 hours, preferably for a period between about 6 and 12 hours, more preferably for about 12 hours. Those skilled in the art will recognize other methods of silylating hydroxyl groups are suitable. The silylating agent may be a silyl trifluoromethanesulfonate in which case the preferred base is pyridine or 2,6-lutidine and the solvent is dichloromethane or chloroform.
Those skilled in the art will recognize that the C-3 hydroxyl of 5,7,3xe2x80x2,4xe2x80x2-tetra-O-benzyl-8-bromoepicatechin or 5,7,3xe2x80x2,4xe2x80x2-tetra-O-benzyl-8-bromoepicatechin can be protected in a similar manner.
Another important step in the present process is the oxidation of the protected epicatechin. The oxidation can be carried out using any suitable oxidant. Typically, the oxidation is carried out in two steps. The first step involves the transformation of the C-4 methylene to a secondary alcohol to provide a protected 4-hydroxyepicatechin, presumably having 4xcex2 stereochemistry. The reaction is typically carried out by contacting a protected epicatechin with an oxidizing agent, preferably a reagent that has been employed to oxidize O-alkylated catechins and epicatechins, such as lead tetraacetate, or 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ). More preferably an electron poor quinone type oxidizing agent such as DDQ or a variation thereof is used. The reaction is carried out in an organic solvent, preferably a dialkyl ether, where the alkyl groups have between 1 and 4 carbon atoms, a cyclic ether such as tetrahydrofuran, or a chlorinated solvent such as dichloromethane or chloroform. When DDQ is the oxidizing agent water is present in the solvent. The reaction is typically carried out at a temperature between 0xc2x0 C. and the reflux temperature of the solvent, preferably at a temperature about 15xc2x0 C. to about 30xc2x0 C., more preferably at about 23xc2x0 C., and for a period of time between about 1 and about 24 hours, preferably about 6 to about 12 hours, more preferably about 12 hours.
The second step is a further oxidation involving the conversion of the protected 4-hydroxyepicatechin to a protected flavan-4-one. The reaction is carried out in the presence of an oxidizing agent, preferably an oxidizing agent formed by combining N-methylmorpholine-N-oxide and tetrapropylammonium perruthenate, and in the presence of a non-reactive drying agent, preferably molecular sieves. This oxidation technique is described by Ley et al. in J. Chem. Soc., Chem. Commun. 1987, 1625. Typically such a reaction is carried out in an organic solvent, preferably a chlorinated alkane such as dichloromethane, at a temperature of between 0xc2x0 C. and the reflux temperature of the solvent, preferably between the temperature of 15xc2x0 C. and 30xc2x0 C., more preferably at a temperature of 23xc2x0 C., and for a period of between 1 and 24 hours, preferably for a period of between 6 and 12 hours, more preferably for a period of 12 hours.
A further transformation in the present process involves the reaction of the protected flavan-4-one with a nucleophilic aryl organometallic reagent to produce a protected 4-aryl-4-hydroxyepicatechin. Typically the reaction is carried out by first forming a nucelophilic aryl organometallic reagent in situ by combining an aryl halide preferably an aryl bromide, with an alkyllithium, preferably with tert-butyllithium, in an anhydrous ether solvent, preferably tetrahydrofuran, under an inert atmosphere, at a temperature of between about xe2x88x92100xc2x0 C. and 0xc2x0 C., for a period of between about 1 and about 24 hours, and then contacting the formed nucleophilic aryl organometallic reagent with the protected flavan-4-one, also in an anhydrous ether solvent, preferably tetrahydrofuran, in an inert atmosphere, at a temperature of between xe2x88x92100xc2x0 C. and 0xc2x0 C., for a period of between about 1 and about 24 hours. Those skilled in the art will recognize that other organometallic reagents such as Grignard reagents may be employed in present invention.
Those skilled in the art will also recognize that certain nucleophilic aryl organometallic reagents may be formed from materials other than aryl halides. In particular nucleophilic aryl organometallic reagents may be prepared by processes known in the art as ortho metallation and transmetallation.
One particular embodiment of the present invention provides for the reaction of 2,4,6-trimethoxyphenyllithium with a protected flavan-4-one. Another particular embodiment of the instant invention contemplates the treatment of protected 8-bromoepicatechin or protected 8-bromoepicatechin or protected 8-bromocatechin with tert-butyllithium to form the nucleophilic organometallic reagent of the invention. A typical protected 8-bromo-epicatechin is 5,7,3xe2x80x2,4xe2x80x2-tetra-O-benzyl-8-bromo-3-O-(tert-butyldimethylsilyl)epicatechin, which is treated with an alkyllithium, preferably tert-butyllithium, in an anhydrous ether solvent, preferably tetrahydrofuran, under an inert atmosphere, at a temperature of between about xe2x88x92100xc2x0 C. and 0xc2x0 C., for a period of between about 1 and about 24 hours, and then contacting the resulting aryllithium with a protected flavan-4-one, also in an anhydrous ether solvent, preferably tetrahydrofuran, in an inert atmosphere, at a temperature of between xe2x88x92100xc2x0 C. and 0xc2x0 C., for a period of between about 1 and about 24 hours.
The process of the present invention provides for the deoxygenation of the protected4-aryl-4-hydroxyepicatechin at C-4 to give a protected 4xcex1-arylepicatechin as a single stereoisomer. The deoxygenation is carried out in the presence of a reducing agent, preferably a reducing agent formed by combining a trialkylsilane in which each alkyl group contains between 1 and 4 carbon atoms, with an organic acid, in an inert organic solvent, preferably a chlorinated alkane such as dichloromethane, at a temperature between 0xc2x0 C. and the reflux temperature for a period between about 5 minutes and about 24 hours. More preferably the reaction is carried out in the presence of a reducing agent formed by combining a trialkyltin hydride in which each alkyl portion contains between 1 and 6 carbon atoms with a perfluorocarboxylic acid, in an inert organic solvent, preferably a chlorinated alkane such as dichloromethane, at a temperature between 0xc2x0 C. and the reflux temperature for a period of between about 5 minutes and about 24 hours.
The invention further provides for a protected 4-aryl-5,7,3xe2x80x2,4xe2x80x2-tetra-O-benzylepicatechin to be deprotected at the C-3 hydroxyl group, or when the 4-aryl substituent is itself an epicatechin, at both C-3 hydroxyl groups. In the embodiment wherein the C-3 hydroxyl group is protected by a silyl group, the deprotection reaction is carried out with fluoride, typically with aqueous hydrofluoric acid in acetonitrile. As those skilled in the art will recognize the C-3 hydroxyl group(s) may be further derivatized after being deprotected. Typically the derivatization will involve acylating the C-3 hydroxyl group with an acylating agent such as an activated acid or acid chloride. A preferred acylating agent is one formed by activating tri-O-benzylgallic acid as an acid chloride. Those skilled in the art will recognize that acids may be activated in a variety of ways to from suitable acylating agents.
Another important step of the process involves the removal of benzyl groups. Benzyl groups may be removed from protected aromatic hydroxyl groups (i.e. phenols) and protected aliphatic hydroxyl groups by hydrogenolysis. Typically, the hydrogenolysis reaction is carried out in an atmosphere of hydrogen gas either at standard temperature and pressure or preferably at elevated pressure of about 1-5 bar, more preferably at about 3.5-5 bar in a suitable organic solvent such as methanol, ethanol, ethyl acetate or a mixture thereof Typically a metal catalyst is used to facilitate the removal of benzyl groups. Preferred catalysts are Pd, Pt, or Ni adsorbed onto a solid support, a more preferred catalyst is palladium hydroxide adsorbed on carbon. However, those skilled in the art will recognize that a variety of other catalyst can be employed to facilitate the removal of benzyl groups. In one embodiment, the removal of the benzyl groups produces a free 4-arylepicatechin which may be isolated and purified. Alternatively, the crude hydrogenolysis product can be directly converted into its acetate derivative, by reacting the crude product with acetic anhydride and a base. In a more preferred embodiment where the 4-aryl substituent is epicatechin, the hydrogenolysis produces a free epicatechin-4xcex1,8-epicatechin and acetylation produces the decaacetate derivative. As these acetate derivatives are shown to be different from their C-4 epimers by 1H NMR spectroscopy, and as the 4,8-position of their interflavan linkage is a necessary consequence of the structure of the starting material, the free epicatechin dimer produced by this process is unequivocally identified as the hitherto unknown epicatechin-4xcex1,8-epicatechin.
In another embodiment, benzyl groups are removed after the C-3 hydroxyl groups have been acylated to produce the free 4-arylepicatechin derivatized at C-3.
The instant invention also provides for the removal of benzyl groups that are introduced as a component of the acylating agent. In a preferred embodiment wherein at least one C-3 hydroxyl group is acylated by an activated derivative of tri-O-benzylgallic acid, the benzyl groups on the gallate hydroxyl groups can be removed in a single step together with the removal of the benzyl groups from the epicatechin core.
The invention is further described by the following non-limiting examples.